To more completely assess the fungal species to which people are exposed, a fungal ribosomal RNA (rRNA) gene sequencing study was designed to test the hypothesis that fungal bioaerosols in the United States indoor built environments are much more diverse than previously estimated using traditional methods of analysis. In FY15, fungal diversity was evaluated in 350 dust samples derived from the bed, bedroom floor and kitchen areas from the homes of asthmatic children and/or mold remediation workers in New York City using Illumina miSeq and compared to results obtained using qPCR. The analysis of fungal diversity in samples derived from these individual studies provided new datasets that have provided greater insight into the fungal species that are frequently recovered in built environments. Analysis of the dust samples showed that Aureobasidium pullulans, Penicillium glabrum, Wallemia sebi and Alternaria alternata varied by housing type (single, multi-family or apartment) and neighborhood asthma prevalence. The preliminary results suggest that multiple environmental factors including anthropogenic behavior, housing type, and neighborhood are important variables that influence fungal diversity within middle-income homes in New York City. A. alternata measured in house dust was associated with fractional exhaled nitric oxide, specifically among children with higher combustion byproduct exposure, suggesting a possible interaction between these two exposures on airway inflammation. As an additional component of this project, NIOSH is comparing the Environmental Relative Moldiness Index (ERMI) to fungal rRNA sequencing data derived from air and dust samples collected from homes with well characterized fungal contamination. The ERMI has been increasingly used by the commercial sector and identifies fungi that typically thrive in moist and wet conditions associated with water infiltration. An expanded dataset will improve the ERMI panel so that it more fully represents the complete spectrum of fungi present in other types of environments. These studies are examining data from a number of different study locations in the United States to categorize and sequence the diversity of fungal rRNA in contaminated and non-contaminated residential and occupational environments. The study locations include homes in Atlanta, Georgia, office buildings in Hartford, Connecticut and Bennington, Vermont, and a school in Wiscasset Maine. A third aspect of this work is the identification and characterization of cross-reactive fungal biomarkers. The overall aim of this portion of the project is to identify broadly cross-reactive fungal allergens with the aim of developing improved immunological methods for detecting fungal bioaerosols and allergic sensitization. To accomplish this, NIOSH is developing species-specific monoclonal antibodies (mAbs) to recombinant fungal antigens. These mAbs will be important for the quantification of species-specific biomarkers of fungal exposure. Each mAb will be tested for reactivity against a panel of closely and distantly related occupational contaminants. MAbs that recognize the same antigen will be selected for further characterization and used in the development of either inhibition or sandwich ELISAs. Aspergillus terreus was initially used as a model fungal organism for these studies. The genome of Chaetomium globosum has now also been sequenced, allowing for the development of fungal-specific proteins using recombinant technologies. Three mAbs that react with the Chaetomium globosum enolase have been developed and characterized. A similar approach is currently being used to identify and characterize additional candidate fungal allergens from Ulocladium chartarum and to develop mAbs that can be used to track exposure and effect. Ulocladium chartarum is a recognized biomarker of moisture infiltration but there are no immunoassays available to detect this species in contaminated environments. The reagents developed in this project will be used to characterize IgE reactivity to cross-reactive allergens shared between allergenic fungal species.